Phase II: Conjugation

1. Phase II: Conjugation • Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance • Results in introduction of polar, ionizable groups to enhance water solubility and hence excretion

2. Major Phase II reactions • Glucuronidation • Sulfation • Conjugation with amino acids • Conjugation with glutathione • Methylation • Acetylation

3. Glucuronidation • Enzyme: glucuronyl transferase, or glucuronosyl transferase • Targets: • hydroxyl groups: Phenols, Alcohols, Dihydrodiols (ether glucuronides) • Carboxylic acids (ester glucuronides) • Amines (N-glucuronides) • Thiols (S-glucuronides) • Carbon (C-glucuronides, rare)

4. Reaction Phenol Phenyl glucuronide

5. Glucuronidation • Conjugating moiety: glucuronic acid, a sugar • Co-factor: UDP-glucuronic acid (UDPGA), derived from glycogen synthesis • Located in endoplasmic reticulum • Multiple families of isoforms:UGT1, UGT2 • UGT1.1 ..1.7, UGT2.1..2.4 • Inducible

6. Uridine-5’-diphospho--D-glucuronic acid (UDPGA)

7. GlucuronidationTypical substrates: • Phenol • 1-Naphthol • 4-Hydroxybiphenyl • 3-Hydroxybenzo[a]pyrene • Benzo[a]pyrene-7,8-dihydrodiol • 2-Naphthylamine • Bilirubin • Steroids

8. Sulfation • Sulfotransferase ST,15 isoforms (xx-ST) • Targets • Hydroxyl groups (phenols, alcohols) • Amino groups • Thiols • Conjugating moiety: sulfuric acid, H2SO4 • Co-factor: 3’phosphoadenosine 5’phosphosulfate (PAPS), formed from ATP + sulfate • Located in cytosol, Probably not inducible

9. SulfationTypical substrates • Ethanol • Phenol • 3-Hydroxybenzo[a]pyrene • Cholesterol • 2-Naphthylamine • N-hydroxy-2-naphthylamine

10. Reaction PAPS PAP

11. Conjugation with amino acids • Amino acid transferases • Targets: carboxylic acids • Conjugating moieties: Glycine, glutamine, alanine, taurine, histidine, ornithine • Co-factor: Acetyl CoA (CoASH) and ATP • In cytosol

12. Reaction Benzoyl-CoA Hippuric acid Benzoic acid

13. Conjugation with glutathione • Glutathione S-transferases (GST) • Targets: Epoxides, halogens • Conjugating moiety: Glutathione • Co-factor: None • Mainly in cytosol • Inducible • Multiple families of isoforms: GSTA, GSTM, GSTP, GSTT ()(αμπθ)

14. Glutathione A tripeptide Glutamic acid (Glu) Glycine (Gly) Cysteine (Cys)

15. Reaction

16. Typical substrates • Organic halides, e.g methyl iodide, benzyl chloride • Alkenes e.g. diethyl maleate • Epoxides

17. Mercapturic acid pathway

18. Methylation • Methyltransferases • Target: Hydroxyl groups, amines, thiols • Substrates mainly endogenous: Catechols, noradrenalin, histamine • Conjugating moiety: Methyl group • Co-factor: S-adenosylmethionine

19. S-adenosylmethionine

21. MethylationReaction Substrate: Catechol Enzyme: Catechol-O-methyltransferase (COMT)

22. Acetylation • N-acetyltransferases (NAT) • Target: Aromatic amines, sulfonamides • Conjugating moiety: Acetyl group • Co-factor: Acetyl-CoA • Few forms: NAT1, NAT2. NAT3: mice • Genetic polymorphisms: “slow and fast acetylators”

23. AcetylationReaction 2-Naphthylamine 2-Aminonaphthalene 2-Acetylaminonaphthalene 2-Acetamidonaphthalene

24. “Other” detoxication mechanisms • P-glycoprotein: ATP-dependent carrier that removes molecules from cells • Multidrug resistance associated protein MDR • Multispecific organic anion transporter MOAT

25. Reactive Oxygen Species (ROS) • Peroxides • Hydrogen peroxide HOOH • Peroxynitrite OONO- • Lipid hydroperoxide LOOH • Free radicals • Superoxide anion O2•- • Hydroxyl radical HO• • Nitric oxide NO•

26. Non-enzymic reaction with anti-oxidants • Ascorbic acid (Vitamin C) • alpha-Tocopherol (Vitamin E) • Glutathione

27. Superoxide dismutase Converts superoxide anions to hydrogen peroxide O2•- +O2•- + 2H+ O2 + H2O2

28. Peroxidases Couple reduction of hydrogen peroxide (or other peroxide) to oxidation of another substrate (co-oxidation) ROOH + R’H ROH + R’OH

29. Peroxidases • Catalase • Prostaglandin synthetase • Myeloperoxidase • Lactoperoxidase • Glutathione peroxidase